Structure of human cortisol-producing cytochrome P450 11B1 bound to the breast cancer drug fadrozole provides insights for drug design.

Human cytochrome P450 11B1 (CYP11B1) is responsible for the final step generating the steroid hormone cortisol, which controls stress and immune responses and glucose homeostasis. CYP11B1 is a promising drug target to manage Cushing's disease, a disorder arising from excessive cortisol production. However, the design of selective inhibitors has been hampered because structural information for CYP11B1 is unavailable and the enzyme has high amino acid sequence identity (93%) to a closely related enzyme, the aldosterone-producing CYP11B2. Here we report the X-ray crystal structure of human CYP11B1 (at 2.1 Å resolution) in complex with fadrozole, a racemic compound normally used to treat breast cancer by inhibiting estrogen-producing CYP19A1. Comparison of fadrozole-bound CYP11B1 with fadrozole-bound CYP11B2 revealed that despite conservation of the active-site residues, the overall structures and active sites had structural rearrangements consistent with distinct protein functions and inhibition. Whereas fadrozole binds to both CYP11B enzymes by coordinating the heme iron, CYP11B2 binds to the R enantiomer of fadrozole, and CYP11B1 binds to the S enantiomer, each with distinct orientations and interactions. These results provide insights into the cross-reactivity of drugs across multiple steroidogenic cytochrome P450 enzymes, provide a structural basis for understanding human steroidogenesis, and pave the way for the design of more selective inhibitors of each human CYP11B enzyme.

Marketed Drugs Can Inhibit Cytochrome P450 27A1, a Potential New Target for Breast Cancer Adjuvant Therapy.

Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification.

Aldosterone synthase inhibitors as promising treatments for mineralocorticoid dependent cardiovascular and renal diseases.

Besides the well-known roles of aldosterone as a mineralocorticoid in regulating homeostasis of electrolytes and volume, recent studies revealed that it is also a potent proinflammation factor inducing reactive oxygen species and up-regulating a panel of fibrosis related genes. Under pathological circumstances, excessive aldosterone is involved in a lot of chronic diseases, including hypertension, cardiac fibrosis, congestive heart failure, ventricular remodeling, and diabetic nephropathy. Therefore, the inhibition of aldosterone synthase (CYP11B2), which is the pivotal enzyme in aldosterone biosynthesis, was proposed as a superior approach. Expected pharmacodynamic effects have been demonstrated in both animal models and clinical trials after the application of CYP11B2 inhibitors. The importance of selectivity over other steroidogenic CYP enzymes, in particular 11ß-hydroxylase (CYP11B1), was also revealed. Recently, much more selective CYP11B2 inhibitors have been reported, which could be promising drug candidates for the treatment of aldosterone related diseases.

Transdifferentiation of differentiated ovary into functional testis by long-term treatment of aromatase inhibitor in Nile tilapia.

Females with differentiated ovary of a gonochoristic fish, Nile tilapia, were masculinized by long-term treatment with an aromatase inhibitor (Fadrozole) in the present study. The reversed gonads developed into functional testes with fertile sperm. The longer the fish experienced sex differentiation, the longer treatment time was needed for successful sex reversal. Furthermore, Fadrozole-induced sex reversal, designated as secondary sex reversal (SSR), was successfully rescued by supplement of exogenous 17ß-estradiol. Gonadal histology, immunohistochemistry, transcriptome, and serum steroid level were analyzed during SSR. The results indicated that spermatogonia were transformed from oogonia or germline stem cell-like cells distributed in germinal epithelium, whereas Leydig and Sertoli cells probably came from the interstitial cells and granulosa cells of the ovarian tissue, respectively. The transdifferentiation of somatic cells, as indicated by the appearance of doublesex- and Mab-3-related transcription factor 1 (pre-Sertoli cells) and cytochrome P450, family 11, subfamily B, polypeptide 2 (pre-Leydig cells)-positive cells in the ovary, provided microniche for the transdifferentiation of germ cells. Decrease of serum 17ß-estradiol was detected earlier than increase of serum 11-ketotestosterone, indicating that decrease of estrogen was the cause, whereas increase of androgen was the consequence of SSR. The sex-reversed gonad displayed more similarity in morphology and histology with a testis, whereas the global gene expression profiles remained closer to the female control. Detailed analysis indicated that transdifferentiation was driven by suppression of female pathway genes and activation of male pathway genes. In short, SSR provides a good model for study of sex reversal in teleosts and for understanding of sex determination and differentiation in nonmammalian vertebrates.

Structural insights into aldosterone synthase substrate specificity and targeted inhibition.

Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (cytochrome P450 11B2, CYP11B2) is the sole enzyme responsible for the production of aldosterone in humans. Blocking of aldosterone synthesis by mediating aldosterone synthase activity is thus a recently emerging pharmacological therapy for hypertension, yet a lack of structural information has limited this approach. Here, we present the crystal structures of human aldosterone synthase in complex with a substrate deoxycorticosterone and an inhibitor fadrozole. The structures reveal a hydrophobic cavity with specific features associated with corticosteroid recognition. The substrate binding mode, along with biochemical data, explains the high 11ß-hydroxylase activity of aldosterone synthase toward both gluco- and mineralocorticoid formation. The low processivity of aldosterone synthase with a high extent of intermediates release might be one of the mechanisms of controlled aldosterone production from deoxycorticosterone. Although the active site pocket is lined by identical residues between CYP11B isoforms, most of the divergent residues that confer additional 18-oxidase activity of aldosterone synthase are located in the I-helix (vicinity of the O(2) activation path) and loops around the H-helix (affecting an egress channel closure required for retaining intermediates in the active site). This intrinsic flexibility is also reflected in isoform-selective inhibitor binding. Fadrozole binds to aldosterone synthase in the R-configuration, using part of the active site cavity pointing toward the egress channel. The structural organization of aldosterone synthase provides critical insights into the molecular mechanism of catalysis and enables rational design of more specific antihypertensive agents.

Synthesis, biological evaluation, and molecular modeling of 1-benzyl-1H-imidazoles as selective inhibitors of aldosterone synthase (CYP11B2).

Reducing aldosterone action is beneficial in various major diseases such as heart failure. Currently, this is achieved with mineralocorticoid receptor antagonists, however, aldosterone synthase (CYP11B2) inhibitors may offer a promising alternative. In this study, we used three-dimensional modeling of CYP11B2 to model the binding modes of the natural substrate 18-hydroxycorticosterone and the recently published CYP11B2 inhibitor R-fadrozole as a rational guide to design 44 structurally simple and achiral 1-benzyl-1H-imidazoles. Their syntheses, in vitro inhibitor potencies, and in silico docking are described. Some promising CYP11B2 inhibitors were identified, with our novel lead MOERAS115 (4-((5-phenyl-1H-imidazol-1-yl)methyl)benzonitrile) displaying an IC(50) for CYP11B2 of 1.7 nM, and a CYP11B2 (versus CYP11B1) selectivity of 16.5, comparable to R-fadrozole (IC(50) for CYP11B2 6.0 nM, selectivity 19.8). Molecular docking of the inhibitors in the models enabled us to generate posthoc hypotheses on their binding modes, providing a valuable basis for future studies and further design of CYP11B2 inhibitors.

Fadrozole reverses cardiac fibrosis in spontaneously hypertensive heart failure rats: discordant enantioselectivity versus reduction of plasma aldosterone.

Reversal of cardiac fibrosis is a major determinant of the salutary effects of mineralocorticoid receptor antagonists in heart failure. Recently, R-fadrozole was coined as an aldosterone biosynthesis inhibitor, offering an appealing alternative to mineralocorticoid receptor antagonists to block aldosterone action. The present study aimed to evaluate the effects of R- and S-fadrozole on plasma aldosterone and urinary aldosterone excretion rate and to compare their effectiveness vs. the mineralocorticoid receptor antagonist potassium canrenoate to reverse established cardiac fibrosis. Male lean spontaneously hypertensive heart failure (SHHF) rats (40 wk) were treated for 8 wk by sc infusions of low (0.24 mg/kg.d) or high (1.2 mg/kg.d) doses of R- or S-fadrozole or by potassium canrenoate via drinking water (7.5 mg/kg.d). At the high dose, plasma aldosterone levels were decreased similarly by R- and S-fadrozole, whereas urinary aldosterone excretion rate was reduced only by S-fadrozole. In contrast, whereas at the high dose, R-fadrozole effectively reversed preexistent left ventricular interstitial fibrosis by 50% (vs. 42% for canrenoate), S-fadrozole was devoid of an antifibrotic effect. The low doses of the fadrozole enantiomers did not change cardiac fibrosis or plasma aldosterone but similarly reduced urinary aldosterone excretion rate. In conclusion, R-fadrozole may possess considerable therapeutic merit because of its potent antifibrotic actions in the heart. However, the observed discordance between the aldosterone-lowering and antifibrotic effects of the fadrozole enantiomers raises some doubt about the mechanism by which R-fadrozole diminishes cardiac collagen and about the generality of the concept of lowering aldosterone levels to treat the diseased heart.

Lead optimization providing a series of flavone derivatives as potent nonsteroidal inhibitors of the cytochrome P450 aromatase enzyme.

Following our SAR studies on aromatase inhibitors, new compounds were designed by appropriately modifying the structure of flavone 1 using our previously reported CoMFA model. While the introduction of substituents on the 2-phenyl ring alone did not cause improvement in potency, these modifications and the removal of the 7-methoxy group led to compounds showing inhibitory activity in the nanomolar range, comparable to the marketed drug fadrozole.

Can the dextroenantiomer of the aromatase inhibitor fadrozole be useful for clinical investigation of aldosterone-synthase inhibition?

The beneficial effects of spironolactone, eplerenone, amiloride and potassium in preventing cardiovascular damage in various experimental models of salt-induced hypertension can be dissociated from blood pressure effects, and have drawn attention to the direct genomic and non-genomic actions of aldosterone at the level of the vessels, the heart and the kidneys. Exposure to endogenous aldosterone could be decreased by direct and specific aldosterone-synthase inhibition. FAD 286A, the dextroenantiomer of the aromatase inhibitor fadrozole, might be a first candidate to investigate in humans, the physiological impact and therapeutic properties of aldosterone-synthase inhibition, especially in various forms of primary aldosteronism.

Three-dimensional model of cytochrome P450 human aromatase.

A three-dimensional (3-D) structure of human aromatase (CYP 19) was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of an eukaryotic cytochrome P450 and was evaluated by docking S-fadrozole and the steroidal competitive inhibitor (19R)-10-thiiranylestr-4-ene-3,17-dione, into the enzyme active site. According to a previous pharmacophoric hypothesis described in the literature, the cyano group of S-fadrozole partially mimics the steroid backbone C(17) carbonyl group of (19R)-10-thiiranylestr-4-ene-3,17-dione, and was oriented in a favorable position for H-bonding with the newly identified positively charged residues Lys 119 and Arg435. In addition, this model is consistent with the recent combined mutagenesis/modeling studies already published concerning the roles ofAsp309 and His480 in the aromatization of the steroid A ring.

Enantioselectivity of some 1-[(benzofuran-2-yl) phenylmethyl] imidazoles as aromatase (P450AROM) inhibitors.

The enantioselectivity ratio ((+)-:(-)-forms) of three substituted 1-[(benzofuran-2-yl) phenylmethyl] imidazoles as inhibitors of aromatase (P450AROM) was 2.16, 12.3 and 1.0 for the 4-methyl-, 4-fluoro- and 4-chloro-substituted compounds, respectively. The (+/-)-compounds were all >1000 times more potent than (+/-)-aminoglutethimide (IC50 = 12 x 10(3) nM). High potency (5.3-65.0 nM) for all the enantiomers studied is unusual since activity usually resides in one form for chiral inhibitors of P450AROM. The 4-methyl derivative was fitted into the model [Furet, P., Batzl, C., Bhatnager, A.S., Francotte, E., Rihs, G. and Lang, M. (1993) J. Med. Chem. 36, pp. 1393-1400] for binding of S-(-)-fadrazole to the active site and the (R)- and (S)- forms both gave a good fitting pattern with (S)-(-)-fadrazole so accounting for their close activity. Docking of both forms into the active site model for P450AROM [Laughton, C.A., Zvelebil, M.J.J.M. and Neidel, S. (1993) J. Steroid Biochem. Mol. Biol. 44, pp. 399-407], using the orientation of (S)-(-)-fadrazole, gave similar strong binding along the position of the C and D rings of the steroid substrate and in the hydrophobic cavity below the A/B rings. The site was probed for group size accommodation using the less potent 4-phenyl analogue (IC50(+/-) = 242 nM): the (S)-form showed restricted access to the region under the A ring due to the extended bulk of the biphenyl group.

Comparative molecular field analysis of non-steroidal aromatase inhibitors: an extended model for two different structural classes.

Aromatase is a cytochrome P450 isozyme, whose inhibition is known to be therapeutically relevant in the treatment of the breast cancer. A comparative molecular field analysis (CoMFA) has been carried out on a series of non-steroidal aromatase inhibitors belonging to two different structural classes. One subset of compounds consists of fadrozole analogues and was studied in a previous work, from which a 'local' 3-D quantitative structure-activity relationship (QSAR) model for the inhibition of aromatase was obtained. In the present paper, that model is extended to include a second subset of compounds bearing a tetralone nucleus and acting at the same enzyme site with the same mechanism as the azoles. The critical alignment step has been solved by using two different steroidal inhibitors of aromatase as rigid templates, on which the non-steroidal compounds have been superimposed. The final 3-D QSAR models are discussed in terms of predictivity and some implications regarding the steric and electronic requirements of steroidal and non-steroidal inhibitors are pointed out.

Design and synthesis of a new type of non steroidal human aromatase inhibitors.

The structure-activity relationship study of one of recently described aromatase inhibitors, compound 1 (MR20814), allowed us to design some related derivatives as potential new inhibitors. Among those we synthesized, chlorophenylpyridylmethylenetetrahydroindolizinone 5 (MR20492) exhibited in vitro a ten-fold higher inhibition of the enzyme (IC50 = 0.2 +/- 0.0 microM and Ki = 10.3 +/- 3.3 nM).

Synthesis and aromatase-inhibitory activity of imidazolyl-1,3,5-triazine derivatives.

Triamino-substituted 1,3,5-triazine derivatives were synthesized and tested for inhibitory activities against the aromatase of human placental microsomes and the cytochrome P450 side chain cleavage of cholesterol (P450SCC) of pig adrenal mitochondria. The compounds having imidazolyl and tertiary amino groups as substituents in the 1,3,5-triazine ring showed significant aromatase-inhibitory activity. Among them, compounds 17, 23, 26, 27 and 28 were more active than the reference compound, CGS 16949A. The inhibitory activities of these compounds against P450SCC were much weaker than their aromatase-inhibitory activities. These compounds may be regarded as selective aromatase inhibitors.

Molecular modelling study of the binding of inhibitors of aromatase to the cytochrome P-450 heme.

A novel molecular modelling study, involving inhibitors bound to a 'substrate-heme complex', is described for steroidal and non-steroidal inhibitors of Aromatase (AR). Results with azole compounds such as CGS-16949A, and its derivatives, agree with recently reported studies that these compounds appear to utilise the steroid C(17) carbonyl binding region of the active site as opposed to the steroid C(3) carbonyl binding region. The study of Aminoglutethimide (AG) type compounds, however, suggests that they mimic the steroid C(17) and not the C(3) carbonyl group as suggested by previous workers. However, results with inhibitors based on pyridine ligands such as 3-(4'-pyridyl)-3-ethyl piperidine-2, 6-dione (PYG), suggest that these compounds utilise the steroid C(3) carbonyl binding region and therefore agrees with previous reports. Consideration of the orientation of the R and S enantiomers of PYG is, however, found to be a reversal of that previously reported. Using inhibitors bound to the 'substrate-heme complex', and data from previous studies of derivatives of androstenedione, reasons for differences in activity of enantiomers of AG, PYG, N-octyl-3-(4'-pyridyl)-3-ethyl piperidine-2, 6-dione, and 10-thiiranylestr-4-ene-3, 17-dione, as well as other potent and less potent inhibitors, are put forward.

Comparative molecular field analysis of non-steroidal aromatase inhibitors related to fadrozole.

A series of non-steroidal inhibitors of aromatase, structurally related to fadrozole (2), was investigated with the aim of developing a 3D QSAR model using the Comparative Molecular Field Analysis (CoMFA) technique. The alignment of the molecules was performed following two approaches (atom-by-atom and field fit), both starting from an initial hypothesis of superimposition of fadrozole to a steroidal inhibitor (3). From a number of CoMFA models built with different characteristics, one was recognized as the most statistically relevant; this one is discussed in detail. The features of the 3D QSAR model are consistent with those of other 3D and QSAR models of aromatase and its inhibitors.

Structure-activity relationships and binding model of novel aromatase inhibitors.

The use of aromatase inhibitors is an established therapy for oestrogen-dependent breast cancer in postmenopausal women. However, the sole commercially available aromatase inhibitor, aminoglutethimide, is not very selective. We have therefore developed fadrozole hydrochloride and CGS 20,267, which are both currently under clinical evaluation. This report will present an analysis of structure-activity relationships in the azole series of inhibitors and give an account of the further optimization of our development compounds, starting from CGS 20,267 over CGP 45,688 and leading to CGP 47,645, the most potent aromatase inhibitor in vivo reported to date. In addition, on the basis of comparisons of these azole-type inhibitors with the most potent steroidal inhibitors published in the literature, we propose a CAMM-generated model describing the relative binding modes of these two classes of compounds at the active site of the enzyme.

Separation of the enantiomers of some racemic nonsteroidal aromatase inhibitors and barbiturates by capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) and micellar electrokinetic capillary chromatography (MECC) were applied to the resolution of racemic nonsteroidal antiaromatase drugs and intermediates. Successful results were obtained in both modes using alpha-cyclodextrin (alpha-CD), beta-cyclodextrin (beta-CD), gamma-cyclodextrin (gamma-CD), or 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CD) as chiral selectors. Depending on the structure of the solute, one of the cyclodextrins was generally better suited for resolution of the racemate. The basic solutes were analyzed under HPCE conditions, whereas the nonionizable compounds such as glutethimide (Doriden) were analyzed in MECC mode. For the azole-type antiaromatase Fadrozole, both HPCE and MECC modes could be used to achieve the separation of the enantiomers. The influence of experimental factors such as pH, the presence of organic modifier, temperature, the micelle concentration, and the concentration of the chiral selector is also discussed on the basis of the results obtained with some chiral barbiturates. The possibility of analyzing the enantiomers directly in plasma samples was also demonstrated.

Structure-activity studies of non-steroidal aromatase inhibitors: the crystal and molecular structures of CGS 16949A and CGS 18320B.

The crystal and molecular structures of 4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-5-yl)benzonitrile hydrochloride (CGS 16949A) and bis(p-cyanophenyl)imidazo-1-yl methane hemisuccinate (CGS 18320B) have been determined as part of structure-activity relationship studies of non-steroidal aromatase inhibitors. CGS 18320B crystallizes with two inhibitor molecules in the asymmetric unit that are similar in conformation. The cyanophenyl groups and the imidazole moieties in the CGS 18320B molecules display a propellor-like arrangement. The orientation of the imidazole ring in CGS 16949A, which is constrained by the piperidine ring, differs by about 80 degrees from the orientations in both CGS 18320B molecules. The conformations of both compounds are consistent with the proposed model (Banting et al. (1988) J. Enz. Inhibit., 2, 216) for inhibitor binding by positioning of the cyanophenyl group in the steroid A-ring binding site and interaction of the imidazole nitrogen with the iron of the haem.